An insulin activity messenger material and its precursor material are disclosed which have been isolated from hapatic plasma membrane cells that have been incubated with an enzyme known as a phosphatidylinositol-specific phospholipase C. The messenger material comprises a carbohydrate-based compound that exhibits the ability to modulate the activity of the insulin-sensitive enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase, and low Km cAMP phosphodiesterase, and thereby effectuate the activity of insulin on the cellular level. The precursor material comprises an inositol containing glycolipid capable of phosphodiesteractic cleavage by a phosphatidylinositol-specific phospholipase C. The enzyme phosphatidylinositol-glycan specific phospholipase C has also been purified and found to participate in many of the activities identified with respect to the messenger and its precursor. Numerous diagnostic and therapeutic utilities are proposed, and testing procedures, materials in kit form and pharmaceutical compositions are likewise set forth.
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1. A insulin activity messenger material comprising at least one carbohydrate-based compound capable of modulating the activity of the enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase, and low Km cAMP phosphodiesterase from fat cells, and thereby effectuating the activity of insulin on the cellular level.
2. The messenger material of
3. The messenger material of
4. The messenger material of
5. The messenger material of
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The research leading to the present invention was funded in part by a grant from the National Institutes of Health and the New York State Health Research Council.
The present application is a continuation-in-part of co-pending application Ser. No. 850,842, filed Apr. 11, 1986 by the inventor herein.
The Applicant is an author or co-author of several articles directed to the general area of the subject matter of the present invention. (1) [Applicant is a co-author with S. Jacobs, M. Siegel, and P. Cuatrecasas] "Insulin Stimulates The Release From Liver Plasma Membranes Of a Chemical Modulator of Pyruvate Dehydrogenase", BIOCHEM. BIOPHYS. RES. COMM., 102 (3) at 1041-1047, (October, 1981); (2) [Applicant, M. Siegel, S. Jacobs and P. Cuatrecasas] "Putative Mediators of Insulin Action: Regulation of Pyruvate Dehydrogenase and Adenylate Cyclase Activities", PROC. NATL. ACAD. SCI. U.S.A., 79: 3513-3517 (1982); (3) [Applicant, A. Doble, S. Jacobs and Cuatrecasas] "Putative Mediators of Insulin Action Regulate Hepatic Acetyl CoA Carboxylase Activity", BIOCHEM. BIOPHYS. RES. COMM., 110 (3) at 789-795, (February, 1983); (4) [Applicant is sole author] "Preparation and Characterization of Putative Insulin Mediators From Liver", METHODS DIAB. RES., Vol. I, Laboratory Methods, Part B, Pages 73-79 (1984), J. Larner and S. Pohl, Eds., John Wiley & Sons Inc; (5) [Applicant is a co-author with J.A. Fox, P. Sherline and P. Cuatrecasas] "Insulin Stimulates the Hydrolysis of a Novel Membrane Glycolipid Causing the Generation of cAMP Phosphodiesterase Modulators", SCIENCE, 233:967-972 (1986); (6) [Applicant is co-author with P. Cuatrecasas] "Insulin Stimulates the Generation of cAMP Phosphodiesterase Modulators from Hepatic Plasma Membranes", PROC. NATL. ACAD. SCI., 83:5793-5797 (1986); (7) [Applicant is sole author] "Insulin Generates an Enzyme Modulator from Hepatic Plasma Membranes: Regulation of cAMP Phosphodiesterase, Pyruvate Dehydrogenase and Adenylate Cyclase", ENDOCRINOLOGY, 120:967-972 (1987); (8) [Applicant is a co-author with P. Sherline and J.A. Fox], "Insulin Stimulated Diacylglycerol Production Results from the Hydrolysis of a Novel Phosphatidylinositol-Glycan", J. BIOL. CHEM., 262:1116-1121 (1987); and (9) [Applicant is a co-author with J.A. Fox and N.M. Soliz] "Purification of a Phosphatidylinositol-Glycan-Specific Phospholipase C From Liver Plasma Membranes; a Possible Target of Insulin Action", (in press). All of the above listed articles are incorporated herein by reference.
The present invention is generally directed to materials and associated methods for the analysis and treatment of diabetic disorders, and in particular, is concerned with the identification, analysis and application of materials that may participate in the transmission and effectuation of insulin action that averts the occurrence of the diabetic condition.
For some time, diabetic research has focused on the action of insulin at the cellular level, and in particular, has determined that insulin action may take place by the generation of intracellular mediators which modulate certain key enzymes. Thus, it has been believed for some time that insulin action at the cellular level is not direct but rather occurs through the stimulation by insulin of its cellular receptors which in turn, cause the generation and dispatch of hitherto chemically undefined substances which stimulate certain key enzyme activities. Thus, while the diabetic condition is largely attributed to an inability of the patient to synthesize sufficient insulin, a particular variety of diabetes known as Type II adult-onset diabetes is recognized, wherein the patient is capable of synthesizing sufficient or near sufficient quantities of insulin and likewise possesses sufficient or near sufficient cellular receptors, however, the activities of insulin are not carried out. Applicant and the co-workers identified in Publication Nos. 1-4 above, have conducted extensive investigations of the cellular environment, in particular with rat liver fractions, and determined that such mediators or messengers exist that appear to modulate the activities of certain key enzymes such as pyruvate dehydrogenase, adenylate cyclase and acetyl CoA carboxylase. Despite the extensive knowledge gained from the review of the results of these investigations, there still existed no knowledge as to the actual mechanisms on the cellular level that were involved in the generation of the messenger material, nor was there a suggestion as to the chemical structure and correspondingly, the identity and number of constituents of such messenger material.
In Publication No. 4, the present inventor speculated that two materials may exist, the first being a stimulator and the second an inhibitor. Notwithstanding efforts to characterize the molecular weight of these materials and to determine within some general scope the activity of these materials, the exact identity of the materials has remained unknown as has their origin and action.
The present application is therefore directed to the identification of the messenger material as to its structure, origin and action, and to corresponding diagnostic and therapeutic applications to which the messenger material may be put.
In accordance with the present invention, a messenger material for insulin activity has been isolated which comprises at least one carbohydrate-based compound capable of modulating the activity of the enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase and low Km cAMP phosphodiesterase from fat and liver cells, and thereby effectuating the activity of insulin on the cellular level. In one embodiment the messenger material is derivable from hepatic tissue, and in particular may be derived from hepatic plasma membranes that have been incubated with an enzyme known as a phosphatidylinositol-specific phospholipase C. Alternately, this enzyme may be incubated with an extract derived from a culture of an insulin-responsive murine myocyte cell line, identified as the cell line BC3 Hl, which becomes responsive to insulin upon differentiation following attainment of confluence.
More particularly, two carbohydrate-containing compounds have been identified in accordance with the present invention that appear to act as messengers. These compounds share certain common characteristics, among them a non-proteinoid structure, a net negative charge believed to be attributable to the presence of a charged substituent such as phosphate or sulphate, relative polarity, and the presence of glucosamine. More specifically, the messenger compounds are believed to comprise, respectively, a 1,2 cyclic phosphodiester inositol derivative, and a 1- or 2-phosphomonoester inositol derivative, each in respective combination or mixture with glucosamine, and other carbohydrate constituents.
The messenger material of the present invention has been found to be produced by the phosphodiesteratic cleavage of an inositol-containing glycolipid precursor material, the discovery of such precursor material also comprising an aspect of the present invention. Accordingly, the precursor material of the present invention is isolatable from a substrate such as hepatic cells and the insulin-responsive murine myocyte cell line, referred to above. The precursor material identified in accordance with the present invention has been determined to comprise a glycolipid containing the carbohydrates inositol and glucosamine, as well as a diacylglycerol. Further elucidation of the precursor material is presently in progress.
Further, the present invention includes a purified form of the enzyme phosphatidylinositol-glycan-specific phospholipase C which catalyzes the hyrolysis of the novel phosphatidylinositol-glycan (the precursor material) to the inositol-phosphate glycan enzyme modulators (the messenger material) and diacylglycerol. This enzyme, which in one instance has been found in the plasma membrane fraction of rat liver, is purified by a process of solubilization with neutral non-ionic detergent, subsequent anion-exchange chromatography on a DEAE-celluse support and further hydrophobic chromatography on butyl agarose. Purification is verified by gel permeation chromatography which reveals a stokes radius of 35 Angstroms and an apparent molecular weight of 62,000.
The enzyme has been found to be specific for glycosylated phosphatidylinositol which indicates that it is an effector for some of the metabolic actions of insulin. As an effector for these actions of insulin, the purified form of the enzyme can be utilized in the same or a similar manner to those for the messenger material or its precursor material. The enzyme can serve the same purpose as the messenger material or its precursor in the diagnostic and therapeutic methods outlined above. Thus, it can be utilized to measure the amount of messenger material or its precursor or the enzyme itself in the condition of diabetes or other hormonal abnormality.
Additionally, the purified form of the enzyme can be utilized in a process for the preparation of the messenger material, thus affording an improvement in the preparation of the messenger material. For example, exogenously derived precursor material could be prepared and then reacted in vitro with the enzyme of the present invention to prepare the messenger materials in commercial quantities. Further, the enzyme itself could be prepared in commericial quantities by clonal replication utilizing known recombinant techniques.
The discovery of the insulin activity messenger material, its precursor material and the purified enzyme offers the opportunity to further investigate the exact mechanism of the action of insulin and possibly other hormones, and to formulate appropriate therapies in the instance where such action is abnormal and diabetic disorders of the type mentioned earlier herein occur. Thus, the messenger material and stable analogs thereof may be pharmacologically useful in the treatment of such disorders. Moreover, the biosynthesis, generation, activities, degradation and processing of the messenger material, the precursor material and the enzyme may be the subject of various diagnostic procedures, and accordingly, all of these materials may be appropriately prepared with detectable labels for use in both in vitro and possibly in vivo diagnostic procedures. For example, radiolabeled quantities of these materials may be introduced into sample fluids and cellular systems on an in vitro basis and observed to determine the course of activity and movement.
The invention further includes a method for detecting diabetes or other hormonal abnormalities in mammals which comprises the measurement of the production, activity or degradation of a target material selected from the group consisting of an insulin messenger material, its precursor or the enzyme, phosphatidylinositol-glycan specific phospholipase C. In particular, hormones or hormonal conditions such as diabetes could be identified and detected by the suppression or other alteration in the activity of the messenger material, its precursor or the aforesaid enzyme upon the target insulin-sensitive enzymes and substrates that they affect. In this method, cells derived for example, from the insulin-sensitive murine myocyte cell line could be inoculated with the messenger material, its precursor material, or the enzyme as a control, while parallel cellular samples could be inoculated with the same quantity of messenger material, precursor material or the enzyme and one or more agents suspected of adversely affecting insulin action. All samples could thereafter be incubated in accordance with the methods described above, and thereafter subjected to the sequence of separation techniques also defined, whereupon testing of the resulting isolates derived from the control and unknown samples could be compared to determine whether the material is adversely affected by observing the effect if any, on the listed target enzymes or substrates.
Thus, the insulin activity messenger material, its precursor material or the enzyme phosphatidylinositol-glycan specific phospholipase C can be measured by:
(a) preparing at least one sample of said insulin activity messenger material, its precursor or the enzyme from a corresponding number of distinct known stimuli;
(b) preparing a corresponding number of antibodies from said messenger material, precursor or enzyme samples;
(c) placing a detectible label on a material selected from the group consisting of said sample and said antibodies thereto;
(d) placing the labeled material from Step (c) in contact with a biological sample from said mammal in which hormonal disorders are suspected; and
(e) examining said biological sample for the presence of said labeled material. The messenger material, its precursor or the enzyme phosphatidylinositol-glycan-specific phospholipase C can also be measured by:
(a) preparing at least one sample of said messenger material, its precursor or the enzyme from a corresponding number of distinct known stimuli;
(b) placing a detectable label on the sample or samples prepared in Step (a);
(c) placing the labeled sample prepared in Step (b) in contact with a biological sample from said mammal in which hormonal disorders are suspected; and
(d) examining the biological sample for the presence of any of the labeled sample.
In similar fashion, an assay system for screening of potential drugs effective to treat diabetic and other possible hormonal abnormalities related to messenger activities, may be prepared. In one instance, the test drug could be administered to an untreated murine myocyte sample to determine its effect upon the production of the messenger material or upon the enzyme. In an alternate procedure, the messenger material or the enzyme may be introduced into the cellular test system, and the prospective drug may then be introduced to the resulting cell culture and the culture may thereafter be examined to observe any changes in the activity of the messenger material, or the enzyme either from the addition of the prospective drug alone, or the effect of added quantities of the known messenger material or enzyme.
The present invention also relates to a method for detecting the presence or onset of diabetic or similar pathology in mammals, by measuring the activity and presence of the messenger material its precursor, or the enzyme of the present invention. Pathological states may result from hormonal insensitivities in which cells are deficient in the biosynthesis of the precursor material, the enzyme responsible for hydrolysis of the precursor, or coupling of the hormone receptor to activation of the enzyme. Alternatively, the hormone-stimulated generation of the messenger may be unaltered, but the activity of the messenger may be diminished by enzymatic modification or degradation. Thus, the generation, activity or metabolism of the messenger material may be followed directly by the assay techniques discussed later on, through the use of an appropriately labeled quantity of the material. Alternately, the messenger or the enzyme can be used to raise binding partners or antibodies that could in turn, be labeled and introduced into an appropriate medium to test for the presence of messenger material or enzyme therein, and to thereby assess the pathological state of the host from which the medium was drawn.
Thus, both the messenger material and the enzyme and any antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the messenger material or the enzyme that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
In an immunoassay, a control quantity of the messenger material, the enzyme or their antibodies, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample of a mammal believed to be suffering from a diabetic or other hormonal disorder. After the labeled material or its binding partner(s) has had an opportunity to react with receptor sites within the sample, the resulting mass may be examined by known detecting techniques, which may vary with the nature of the label attached.
In the instance where a radioactive label, such as the isotopes 14 C, 131 I, 3 H, 125 I and 35 S are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric or gasometric techniques known in the art.
The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of messenger material or enzyme activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the messenger material or the enzyme; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
Furthermore, the invention includes an assay system to quantitate the possible degradative activity present in tissues, cells or extacts thereof in patients with hormonal disorders. This system or test kit may comprise a labeled compound prepared as detailed above, with one or more chemical or immunological reagents or instructions on chromatographic degradative or modified products of the messenger substance.
In a further embodiment, the present invention relates to therapeutic methods of treatment for diabetic disorders, such as insulin resistance, or altered metabolism in mammals, by the administration of a physiologically effective amount of the enzyme, the precursor material, or the messenger material, or analogs synthesized according to information derived from the structure activity experiments of the present invention. This method of treatment is accomplished by utilizing a pharmaceutical composition comprising a physiologically effective amount of a material selected from the group consisting of the enzyme, the precursor material or an insulin activity messenger material and analogs thereof; and a pharmaceutically suitable carrier therefor.
Thus, in the instance where type II adult-onset diabetes is present, the condition of the patient may be monitored and appropriate of the materials of the present invention may be administered on a periodic basis to supplement the insulin activity system of the patient and to thereby counteract the adverse effects of a messenger or precursor deficiency. Correspondingly, in instances where insulin activity is excessive due to altered metabolism in humans, appropriate antibodies to the enzyme, the precursor material and messenger material and analogs thereof may be developed and administered in a physiologically effective amount along with a pharmaceutically acceptable carrier therefor to inhibit the action of these materials and to thereby control the insulin activity system of the patient to correct any abnormalities thereof.
Accordingly, it is a principal object of the present invention to provide a method for the preparation of an insulin activity messenger material or analog that participates in effectuating insulin activity on the cellular level.
It is a further object of the present invention to provide a method for the preparation, identification and isolation of a precursor material for the insulin activity messenger material.
It is a further object of the present invention to provide a method for the preparation and purification of the enzyme phosphatidylinosital-glycan-specific phospholipase C.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in combating the adverse effects resulting from the lack of the insulin activity messenger material within the proper amounts required for normal cellular insulin activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to avert the onset of diabetic disorder caused by the lack of the proper quantity and activity of the insulin activity messenger material of the present invention.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods relating to the regulation of the quantity of the insulin activity messenger material.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.
FIG. 1--QAE-Sephadex Chromatography of PDE-Modulating Activities. Partially purified extracts from liver membranes were loaded onto a 10 ml QAE-Sephadex column, equilibrated in 0.05M TEA-formate, pH 4.75. The column was eluted with a 40 ml linear gradient of 0.05-0.50M TEA-formate, pH 4.75 as shown: 2 ml fractions were lyophilized, resuspended in 0.5 ml 10 mM formic acid and assayed at a 1:10 dilution for ability to stimulate cAMP phosphodiesterase in adipocyte plasma membranes.
FIG. 2--Analytical SAX HPLC of PDE-Modulating Activities. Peaks I and II from QAE-Sephadex were separately pooled and injected onto an analytical SAX HPLC column, equilibrated with 60% methanol and eluted at 1 ml/min with a linear, 15 min gradient to 0.5M TEA-formate, pH 3.75. One ml fractions were lyophilized, resuspended in 0.5 ml 10 mM formic acid and assayed for PDE-modulating activity.
FIG. 3--P-2 Gel Filtration of PDE-Modulating Activities. Pooled active fractions from analytical SAX were loaded onto a 25 ml P-2 column and eluted with 50 mM formic acid. One ml fractions were lyophilized, resuspended in 0.5 ml 10 mM formic acid and assayed for PDE-modulating activity.
FIG. 4--High Voltage Thin Layer Electrophoresis. Purified PDE modulators were spotted on cellulose thin layer plates and electrophoresed at 500 volts for 1 hr. at pH 3.5 (pyridine/glacial acetic acid/H2 O (1:10:189)) or pH 1.9 (88% formic acid/glacial acetic acid/H2 O (50:56:1794)). One cm regions were scraped, eluted with 50% methanol in 10 mM formic acid, evaporated to dryness, resuspended in 0.05 ml 10 mM formic acid and assayed. Inositol monophosphate migrated towards the top of the plate.
FIG. 5--Thin Layer Chromatography of Precursor Glycolipid. Organic extracts of liver membranes were streaked onto an oxalate impregnated silica gel G plate, which was twice developed in chloroform/acetone/methanol/glacial acetic acid/H2 O (10:4:2:21). One cm regions were scraped and eluted with methanol. Lipids were dried under N2, resuspended in 1 ml 50 mM ammonium bicarbonate, pH 7.4 and treated with PI-PLC for 30 min. PDE-modulators were extracted as detailed in Example I and assayed.
FIG. 6--Dose Dependence of PDE Modulation. The activity of cAMP phosphodiesterase was evaluated as a function of concentration of modulator. Purified Peak I or II was added to adipocyte particulate frction of designated dilution and PDE activity was assayed.
FIG. 7--SAX HPLC of Radiolabeled Mediators from Insulin Treated Cells. BC3 Hl cells were cultured at 37°C in Dulbecco's Minimal Essential Medium in the presence of 20% NU serum (Collaborative Research) on collagen coated 10 mm miniwells. Cells (1×106 /well) were preincubated with 10 uCi (A) [2,63 H]myoinositol or (B) [1,63 H]glucosamine (New England Nuclear) for 20 hours in the presence of 20% Nu Serum. Insulin was added to cells in monolayer in serum free media at a final concentration of 10 nM. Reactions were terminated by removal of media and addition of 1 ml chloroform/methanol/1N HCl (200:100:1), followed by addition of 0.5 ml 10 mM formic acid. Organic and aqueous phases were separated by centrifugation at 500 x g for 5 min.
The water soluble labeled products were purified by a modification of a previously described protocol for purification of cAMP phosphodiesterase (PDE) modulators. The aqueous phases were chromatographed on DEAE-Cellulose, eluted with 0.25 TEA-formate, pH 3.75. This fraction was eluted through a C-18 reversed phase sepak in the same buffer. Following lyophilization, the solution was eluted through Dowex 50WX-4 in 50 mM TEA-formate. pH 3∅ After exposure to activated charcoal, samples were chromatographed on an SAX HLPC column (Whatman), eluted with a linear, 15 minute gradient of 60% methanol (A) to 0.5M TEA-formate, pH 3.75 (B) at 1 ml/minute. One ml fractions were counted. The two peaks of insulin stimulated radioactivity coeluted with similarly purified, myocyte derived PDE modulating activities.
FIG. 8--SAX HPLC of Radiolabeled Mediators Produced by PI-PLC. Cells were prelabeled as described in FIG. 7, extracted in chloroform/methanol/1N HCl (200:100:1) and centrifuged at 1000 x g for 10 min. The supernatant was dried under N2 and resuspended in 1 mil of 50 mM ammonium bicarbonate, pH 7.4. These solutions were treated with 0.1 ug/ml PI-PLC from S. aureus for 30 min. at 37°C Reactions were terminated by reextraction with 1 ml of chloroform/methanol/1N HCl (200:100:1) and aqueous and organic phases separated. The aqueous phases were then purified as described in FIG. 7 and chromatographed on SAX HPLC column, eluted as described.
FIG. 9--Thin Layer Chromatography of the Radiolabeled Precursor from Insulin Treated Cells. (A) [3 H]inositol and B) [3 H]glucosamine labeled cells were treated with or without 10 nM insulin. Following extraction and phase separation, the organic phase were dried under N2, resuspended in chloroform/methanol/H2 O (9:7:2); and spotted on oxalate impregnated Silica gel G plates. These were twice developed in chloroform/acetone/methanol/glacial acetic acid/H2 O (10:4:2:2:1). One cm regions were scraped and radioactivity determined by scintillation counting. Phosphoinositides were identified by iodine staining of standards.
FIG. 10--Thin Layer Chromatography of the Radiolabeled Precursor After PI-PLC Treatment. Cells were labeled, extracted, treated with or without PI-PLC and reextracted as described in FIG. 8. Organic phases were chromatographed on thin layer plates as detailed in FIG. 9.
FIG. 11--Time Course of Precursor Hydrolysis by Insulin. Cells prelabeled with [3 H]inositol were treated with 10 nM insulin for the designated intervals. Cells were extracted and phase separated as detailed in FIG. 7. Aqueous phases were chromatographed up to and including the SAX HPLC step (upper panel). Results reflect counts from a combination of Peaks I and II. The ratio of counts in these peaks were unchanged over the time course. The precursor residing in the organic phases was identified by the peak on TLC with an Rf =0.22, corresponding to the species depleted by PI-PLC (FIG. 10).
FIG. 12--Time Course of Diacylglycerol and Phosphatidic Acid Production in Response to Insulin. Myocytes were incubated for 20 hrs. in serum free DMEM with [9,103 H]myristic acid (New England Nuclear), complexed 1:1 to bovine serum albumin. Insulin was added to cells in fresh media for the designated intervals, and reactions were stopped by removal of media and addition of 1 ml chloroform/methanol/1N HCl (200:100:1). 0.6ml H2 O was added and upper aqueous phases were discarded following centrifugation at 500×g for 5 minutes. The lower, organic phases were dried under N2 and resuspended in 1 ml diethyl ether followed by addition of 1 ml mM formic acid. The upper ether phase was aspirated and the lower phase reextracted with 1 ml diethyl ether. The combined ether phases, containing diacylglycerol, were dried under N2, resuspended in chloroform and spotted on silica gel G plates which were preactivated at 60°C for 1 hr. Plates were twice developed in petroleum ether/diethyl ether/glacial acetic acid (70:30:2). Five ug of unlabeled dimyristoylglycerol were added to each sample. Spots were visualized by iodine staining. To the lower aqueous phases from the ether extraction were added 1 ml of chloroform/methanol (2:1). Following centrifugation, the resulting upper aqueous phases were discarded and the lower organic phases dried under N2, resuspended in chloroform/methanol/H2 O (9:7:2) and spotted on "soft plus" silica gel plates. These were developed in chloroform/pyridine/70% formic acid (50:30:7), and phosphatidic acid was identified by an iodine stained standard. Lipids were scraped and radioactivity determined in a liquid scintillation counter.
FIG. 13--Gel Filtration of Radioactive and Bioactive Mediators. (A) [3 H]Inositol and B) [3 H]glycosamine labeled substances from Peak I from SAX HPLC were chromatographed on a 45 ml P-2 gel filtration column in 50 mM formic acid. Unlabeled cells were extracted by an identical procedure, and chromatographed on an SAX HPLC column, as described in FIG. 7. Active fractions eluting in Peaks I and II were identified by stimulation of the low km cAMP phosphodiesterase (PDE) activity. This was assayed in adipocyte particulate fractions. Bioactivity in Peak I was chromatographed on the P-2 column (panel C). Fractions were collected and counted or assayed for PDE modulating activity. Radioactivity residing in Peak II from SAX exhibited identical elution volumes on this column (not shown).
FIG. 14--High Voltage Electrophoresis of Radioactive and Bioactive Mediators. After gel filtration, purified radioactive and bioactive fractions residing in SAX Peak I were subject to high voltage electrophoresis on cellulose coated thin layer plates at 500 volts for 1 hr. The pH of the running buffer was 3.5 (pyridine/glacial acetic acid/H2 (1:10:189). One cm regions were scraped and eluted with 50% methanol of 10 mM formic acid. [3 H]Inositol and [3H]glucosamine labeled samples were counted. Bioactive samples were assayed for PDE modulating activity as detailed in FIG. 13. Radioactive and bioactive samples exhibited identical migrations when electrophoresed at pH 1.9 (88% formic acid/glacial acetic acid/H2 O (50:56:1794). Radioactivity and bioactivity residing in Peak II similarly migrated slightly farther from the origin at both pH 1.9 and 3.5, as described above.
FIG. 15--Model for Insulin Stimulation of a Phosphoinositide Glycan-Specific Phosphidipase C. Some of the actions of insulin may be mediated by stimulation of the phosphodiesteratic hydrolysis of a novel phosphatidylinositol containing glycolipid. The binding of insulin to its receptor is linked, perhaps through a coupling protein, to the activation of a phospholipase C which selectively hydrolyses a PI-glycan containing glucosamine and other carbohydrates. This hydrolysis results in the generation of two potential signals: (1) an inosito-phosphate glycan messenger activity which regulates cAMP phosphodiesterase and perhaps other insulin sensitive enzymes, and (2) diacylglycerol which may selectively regulate the activity of protein kinase C or an analogous protein kinase.
FIG. 16--DEAE-Cellulose (DE-52) Chromatography of PI-glycan PLC. The supernatant fraction of the solubilized rat liver plasma membranes was applied to a 20 ml DE-52 column (2.5 cm×5 cm) equilibrated in 1% NP-40, 25 mM Tris-HCl, pH 8.0 at 0°C PI-glycan PLC was eluted with 50 mM NaCl in 0.5% NP-40, 25 mM Tris-HCl, pH 8.0 at 0°C at a flow rate of 20 ml/hr. All buffers contained 10 ug/ml each of leupeptin, aprotinin and chymostatin. Enzyme activity was localized by assay with [3 H]C-terminal glycopeptide of mfVSG for 1 hr at 37°C, followed by toluene extraction and analysis by TLC as described in Example V. The arrow refers to where elution with 50mM NaCl was begun. Dpm refers to counts of DAG released.
FIG. 17--Butyl-Agarose Chromatography of PI-glycan PLC. PI-glycan PLC from DE-52 was passed through a column of Extractigel D of equivalent volume to remove detergent and adsorbed onto a 10 ml butyl-agarose column (1.5 cm x 6 cm) equilibrated in 25 mM Tris pH 8.0 at 0°C The column was washed with 20 of the same buffer and then PI-glycan PLC activity was eluted with 0.1% NP-40, 25 mM Tris, pH 8.0 at 0°C at a flow rate of 20 ml/hr. Enzyme activity was identified by assay for 1 hr with C-terminal glycopeptide of mfVSG as described in Example V. Dpm refers to counts present in the organic phase following toluene extraction of the incubation mixture.
FIG. 18--NaDodSO4 -PAGE of PI-glycan PLC. Samples from each stage of the purification were electrophoresed on 10% polyacrylamide gels. Lanes: (1) Solubilized plasma membranes, 25 ug, (2) Supernatant after centrifugation of solubilized plasma membranes, 25 ug, (3) PI-glycan PLC after DE-52 chromatography, 25 ug, (4) PI-glycan PLC after butyl-agarose chromatography, 25 ug. The enzyme in lane 4 was incubated on ice with 10% TCA for 1 hr. Following centrifugation in an Eppendorf microfuge for 0.5 hr., the pellet was washed twice with cold ether and dissolved by boiling in sample buffer. Protein was visualized by silver staining with the Bio-Rad kit. Molecular weight markers were from Sigma.
FIG. 19--AcA 44 Molecular Sizing of PI-Glycan PLC. A 50 ml AcA 44 sizing column was equilibrated in 0.5% Nonidet P-40, 20% glycerol, 25 mM Tris-HCl, pH 8.0 at 0°C Activity was localized by assay with C-terminal glycopeptide of mfVSG. The flow rate of the column was 3 ml/hr. inset: The column was standardized with myosin, phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase.
FIGS. 20 and 21--Product Analysis of PI-glycan PLC Catalyzed Hydrolysis of [3H]myristate-labeled substrates. Butyl-agarose purified PI-glycan PLC was incubated with (A) mfVSG (B) C-terminal glycopeptide for 2 hrs. as described in Example V. The incubation mixtures were twice extracted with chloroform:methanol and analyzed by TLC. DAG was localized both by iodine staining of dimyristylglycerol standard and by [3 H]-DAG produced by trypanosomal phospholipase C.
FIG. 22--Product Analysis of PI-glycan PLC Catalyzed Hydrolysis of [3 H]inositol-labeled PI-glycan purified from BC3 Hl cells as described in Example IV, Infra. After a 2 hr. incubation in 0.25 ml final volume, 1 ml of chloroform:methanol:HCl (1N) (200:100:1) was added, followed by 0.5 ml 10 mM formic acid. Solutions were centrifuged at 500×g for 5 min., and the upper aqueous phase was directly injected onto a SAX HPLC column and eluted. Resulting fractions were analyzed by liquid scintillation counting ( ) control, ( ) enzyme.
In its primary aspect, the present invention relates to the isolation and identification of particular factors, referred to hereinafter as the insulin activity messenger material, its precursor material and the purified enzyme phosphatidylinositol-glycan-specific phospholipase C, that have been found to be present on the cellular level as a result of the interaction between insulin and its receptors. In particular, the messenger, precursor and enzyme materials that have been identified and purified to the end extent set forth in accordance with the present invention, represent materials that apparently exist on the cellular level and that are activated modified as a result of receptor binding of insulin to act on behalf of insulin to effectuate insulin activity on the cellular level.
In particular, and as will be more fully set forth hereinafter, it has been determined that receptor binding of insulin promotes the phosphodiesteratic cleavage of the precursor material by the enzyme, which results in the formation of the insulin activity messenger material which then exerts its effects upon the cellular system, as exemplified by its effects shown hereinafter on the enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase and low Km cAMP phosphodiesterase that is derived from fat or liver cells. The activity of the messenger material with respect to all of these enzymes is reflective of activity directly resulting from the presence and regulatory effect of insulin on the cellular level.
Although the focus of considerable research attention over the past two decades, the molecular mechanisms of insulin action remain largely unexplained. Insulin binds to a heterodimeric cell surface receptor which contains a hormonally responsive tyrosine kinase activity in its subunit. The ensuing changes in cellular metabolism are diverse, occurring within seconds or hours of hormone-receptor interaction. The acute regulation by insulin of enzymes controlling intermediary metabolism is observed within minutes, and is often the result of changes in the phosphorylation state of the enzyme. The precise biochemical link between the activated receptor and enzyme regulation, however, remains unknown. Several known substances have been proposed to act as second messengers for insulin, yet each has proven inadequate to account for the action of the hormone. More recent reports have suggested that some of the actions of insulin may be accounted for by the generation from the plasma membrane of a unique undefined substance or group of substances which acutely regulate certain insulin sensitive enzymes, perhaps through control of protein phosphorylation. These mediator substances were water soluble, non-nucleotide and exhibited an apparent molecular weight of 1-2000. Although there was suggestion of a peptidic structure, definitive information concerning the chemical identities of these substances has not been forthcoming.
In accordance with the present invention, two such substances, which modulate the activity of the high affinity cAMP phosphodiesterase (PDE) have been purified from hepatic plasma membranes. These two substances have similar properties and activities, but can be resolved by ion exchange chromatography and high voltage electrophoresis. Both exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximatel 1400 daltons. The production of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol (PI) specific phospholipase C purified from S. aureus. This enzyme is known to selectively hydrolyze PI and release from membranes several proteins which are covalently linked to PI via a glycan anchor. Both PDE-modulating substances appear to generated from a novel phosphatidylinositol glycolipid precursor which has been identified by thin layer chromatography. Some of the chemical properties of these substances have been examined, indicating a complex carbohydrate-phosphate containing glucosamine and inositol.
Thus, the present invention comprises the identification and preparation of an insulin activity messenger which comprises at least one and possibly two carbohydrate-based compounds that have been found to be capable of modulating the activity of the enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase and low Km cAMP phosphodiesterase from fat cells, and thereby effectuating the activity of insulin on the cellular level. The messenger materials may be prepared from cells known to contain a precursor material for said messenger material such as animal hepatic cells, by incubating said cells, or a portion thereof with the enzyme of the present invention, a phosphatidylinositol-specific phospholipase C (hereinafter PI-PLC), and inducing said cells to produce said messenger material as indicated in the above discussion. While the hepatic plasma membrane cells have shown particular activity in this regard, other tissues have been similarly investigated and found to offer comparable results. Also, the insulin-responsive murine myocyte cell line BC3 Hl provides a particularly useful synthetic medium for the in vitro simulation of the activities identified herein, which likewise may serve capably in subsequent assay testing and antibody and drug development activities.
As stated earlier, the insulin activity messenger of the present invention comprises at least one complex carbohydrate having at least one phosphate substituent present as charged group and including a glucosamine component. As will be clear from a review of the examples that follow herein, this data was derived from the purification procedures in Example I. In particular, the complex carbohydrate may be selected from cyclic 1,2 phosphodiester inositol derivatives and a 1- or a 2-phosphomonoester inositol derivative or mixtures of these, each in combination with a glucosamine component. More particularly, and as a result of the analysis of the data generated and set forth herein, it has been determined that the messenger material of the present invention comprises two compounds, the first compound comprising a mixture of a cyclic 1, 2 phosphodiesterinositol derivative and glucosamine, the second compound comprising a mixture of either a 1- or a 2-phosphomonoesterinositol derivative, or a mixture thereof, in combination with glucosamine.
As mentioned earlier, the present invention also includes a precursor material from which the insulin activity messenger material may be produced by the phosphodiesteratic cleavage of an inositol-containing glycolipid precursor catalyzed by a phosphatidylinositol-specific phospholipase C. In particular, and as set forth herein, the precursor material has been identified to the extent that it contains diacylglycerol, inositol and glucsoamine components. In other respects, however, this precursor material is chemically undefined.
The present invention further includes the preparation and purification of the enzyme phosphatidylinositol-glycanspecific phospholipase C. In particular, this enzyme is characterized by the following:
(a) it catalyzes the hydrolysis of glycosylated-PI sustrates which leaves the phosphodiester bond and releases diacylglycerol;
(b) it exhibits no activity in hydrolysis of PI, PI-4,5-biphosphate, or phosphatidylcholine;
(c) it does not require the presence of calcium for activity;
(d) it is stimulated by treatment with the chelator EGTA;
(e) it is activated by exposure to 2-mercaptoethanol;
(f) it is inhibited by mercurial compounds;
(g) enzyme activity that is stabilized by glycerol and ethylene glycol, but is destroyed with repeated freeze-thawing; and
(h) an apparent molecular size of 52,000 daltons with a stokes radium of approximately 35 angstroms.
Further, this embodiment of the invention includes a process for the purification of the enzyme which comprises conducting a detergent solubilization of rat liver plasma membranes, thereafter conducting chromatographic separation of the solubilized membranes on a DEAE-cellulose anion exchange column. Subsequently, the eluate is subjected to further chromatographic separation to remove the detergent fraction and a final chromatographic separation is then conducted on a butyl-agarose column to complete the instant purification.
As mentioned earlier herein, the enzyme of the present invention has numerous utilities corresponding in many respects to those utilities and applications noted respecting the messenger materials. Thus, measurement of the level and activity of the enzyme can likewise be used to determine the status and existence of hormonal disorders such as diabetic conditions. Also, the enzyme may be utilized to prepare messenger materials on an in vitro basis and may itself be prepared by clonal replication if it is desired to develop commercial quantities thereof.
The present invention also relates to a variety of diagnostic applications, including methods for detecting the onset or presence of insulin sensitivity disorders by measuring the activity of the messenger material, its precursor material or the enzyme. As mentioned earlier, the messenger material or the enzyme can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as a test for the presence an activity of the messenger material in suspect mammalian hosts. Thus produced are antibodies to an insulin activity messenger material present in the serum of a mammal, the messenger material to which said antibody is raised which comprises at least one-carboydrate-based compound that has been found to be capable of modulating the activity of the enzymes pyruvate dehydrogenase, adenylate cyclase, acetyl CoA carboxylase, and low Km cAMP phosphodiesterase and thereby effectuating the activity of insulin on the cellular level.
Antibody(ies) to the messenger material or the enzyme can be produced and isolated by standard methods including the well known hybridoma techniques. The antibody(ies) can be used in another species as though they were antigen(s) to raise antibody(ies). Both types of antibody(ies) can be used to determine the presence and extent of activity of the messenger material, its precursor or the enzyme. For convenience, the antibody(ies) to the messenger material, precursor or enzyme will be referred to herein as Ab1 and antibody(ies) raised in another species as Ab2.
Particularly useful antibodies are those which are isolated from the serum of a mammal which suffers from a diabetic disorder.
Abnormalities in the insulin-sensitive production of messenger material activity in various diabetic disorders can be ascertained by the usual immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the messenger material, its precursor or the enzyme labeled with a detectable label, antibody Ab1 labeled with a detectable label, or antibody Ab2 labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "Mes" stands for the messenger, its precursor or the enzyme material:
Mes*+Ab1 =Mes*Ab1 A.
Mes+Ab1 * =MesAb1 * B.
Mes+Ab1 +Ab2* =MesAb1 Ab2 * C.
The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A, is described in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the "sandwich" procedure, is described in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody", or "DASP" procedure.
In each instance, the messenger material, the precursor, or the enzyme forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
It will be seen from the above, that a characteristic property of Ab2 is that it will react with Ab1. This is because Ab1 raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab2. For example. Ab2 may be raised in goats using Ab1 as an antigen. Ab2 therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims, Ab1 will be referred to as a messenger material antibody and Ab2 will be referred to an an antibody reactive with a messenger material antibody or, in the alternative, an "anti-antibody".
The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
A number of fluorescent chemicals are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
The messenger material, its precursor or the enzyme, or their binding partners can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 14 C, 131 I, 3 H, 125 I and 35 S.
Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, urease, glucose oxidase plus peroxidase, galactose oxidase plus peroxidase and acid phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
Purified preparations of messenger material or enzyme added exogenously can regulate the metabolism of intact cells. This suggests the possiblity of a receptor-mediated transport system which acts to internalize the substance into the cell.
A particular assay system developed and utilized in accordance with the present invention, is known as a receptor assay. In a receptor assay, the material to be assayed is appropriately labeled and then certain cellular test colonies such as the adipocyte or myocyte cell system are inoculated with a quantity of the labeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in activity between materials can be ascertained.
In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of messenger material activity in cells or cell extracts derived from patients with obesity or hormonal disorders. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled messenger material, precursor or enzyme or the binding partner thereto, and an antibody specific thereto. Another will contain at least Ab1 together with labeled Ab2. Still another will contain at least Ab1 and directions, of course, depending upon the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.
Accordingly, a test kit may be prepared for the detection of the messenger, its precursor or the enzyme, comprising:
(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the messenger material, its precursor or the enzyme thereto or a specific binding partner thereto, to a detectable label;
(b) other reagents; and
(c) directions for use of said kit.
The test kit may utilize any of the aforementioned detectable lables, i.e., enzymes, radioactive elements, chemicals which fluoresce when exposed to ultraviolet light and others.
More specifically, the diagnostic test kit may comprise:
(a) a known amount of the messenger material as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each;
(b) if necessary, other reagents; and
(c) directions for use of said test kit.
In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive", "sandwich", "double antibody", etc.), and comprises:
(a) a labeled component which has been obtained by coupling the messenger material to a detectable label;
(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:
(i) a ligand capable of binding with the labeled component (a);
(ii) a ligand capable of binding with a binding partner of the labeled component (a);
(iii) a ligand capable of binding with at least one of the component(s) to be determined; and
(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and
(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the messenger material and a specific binding partner thereto.
In accordance with the above, an assay system for screening potential drugs and other agents for the ability to mimic, counteract, block production of or potentiate the messenger material, its precursor material or the enzyme may be prepared. In a first procedure, the test drug could could be administered to an observable hormonally sensitive cell to determine its effect upon cellular metabolism. In an alternate procedure, the messenger material, its precursor material or the enzyme may be introduced into a hormonally sensitive cellular test system such as the adipocyte or myocyte BC3 Hl and the prospective drug may then be introduced into the resulting hormonally sensitive cell culture by inoculation, and the culture thereafter examined to observe any changes in the activity of the messenger material, either from the addition of the prospective drug alone, or the effect of added quantities of the known messenger material. Moreover, the ability of a test drug to prevent or stimulate the production of the messenger material could be screened.
Thus, the activity of the messenger material, its precursor or the enzyme with exposure to hormones can be measured by:
(a) preparing the messenger material, its precursor or the enzyme measured from a hormonally treated cell;
(b) forming a labeled material by placing a detectible label on any of the three materials;
(c) isolating a biological sample selected from the group consisting of blood, tissue and body fluid, from a mammal suspected of hormonal disorders;
(d) placing said material in contact with said biological sample; and
(e) examining said biological sample to locate said labeled material, to thereby determine the activity of material selected for observation.
This method can further comprise preparing antibodies which react with the messenger material, its precursor, or the enzyme which antibodies may be used in said method in the same manner as said messenger material, precursor or enzyme.
A further method is that wherein a second generation of antibodies reactive with the antibodies to the messenger material, its precursor or the enzyme, are prepared which may be used in said method in the same manner as said target material and the antibodies thereto. One of said antibodies reactive with said messenger material or its precursor may be included in said labeled material and one of said antibodies may have placed on it a label. One member of said second generation of antibodies can be included in said labeled material. One member of said second generation of antibodies can be placed on it said label.
As indicated earlier, the following examples set forth the details of the isolation and identification of the present messenger material, its precursor and the enzyme associated with its production, and the observations noted as to their activities, defining both the distinctions and similarities in activity between the present messenger material and those factors identified earlier both by applicant and by others in the field. Naturally, the specific materials and techniques set forth hereinafter are exemplary only and may vary, so that the following is presented as illustrative but not restrictive of the present invention.
PAC Experimental ProceduresAll reagents were from Sigma, with the exception of collagenase (Millipore), DEAE-Cellulose and SAX HPLC columns (Whatman), C-18 reverse phase resin (Waters), P-2 gel filtration media, Dowex AG-1X8 and Dowex 50WX-4 (BioRad), QAE Sephadex (Pharmacia) and 2,8[3 H]cAMP (NEN). Male rats (100-125 g) were from Sprague-Dawley. Monocomponent bovine insulin was from Wellcome Laboratores, Beckenham, U.K. Purified phosphatidylinositol specific phospholipase C (PI-PLC) from S. aureus was a generous gift from Dr. M. Low of the Oklahoma Medical Research Foundation.
These were generated from bovine liver particulate fractions by a modification of previously described methods. Particulate fraction was prepared from 100 g of bovine liver, and suspended in 500 ml mM ammonium bicarbonate, pH 7.4. (Buffer A) at a final concentration of 2-4 mg protein/ml. This solution was incubated with 10mM for 10 min at 37° C., acidified to pH 4.0 with formic acid and sedimented at 45,000 g for 20 min. An equal volume of chloroform/methanol (2:1) was added to the supernatant, and the upper phase was aspirated and methanol was evaporated.
Extracted lipids were treated with 1.0 ug/ml S. aureus PI-PLC for 30 min at 37°C in buffer A. Following incubation, samples were acidified to pH 4.0 and PDE-messengers were extracted with chloroform/methanol as described above.
The low Km cAMP phosphodiesterase (PDE) from rat adipocyte particulate fraction was assayed. Rat fat cells were fractionated on discontinuous sucrose density gradients as previously described. cAMP PDE was measured for 10 min. at 30°C by the method of Thompson and Appleman as modified previously. Only the high affinity enzyme was present in this subcellular fraction. Activity was proportional to protein concentration and linear throughout 30 min. Results are means of triplicate determinations in which variability was less than 5%. Protein concentration was measured by the method of Lowry et al.
Purified mediator fractions were subject to high voltage electrophoresis on cellulose coated thin layer plates. Plates were subject to 500 volts for 1 hr. at pH 3.5 or 1.9. 1 cm regions were scraped, eluted with 50% methanol in 10 mM formic acid, lyophilized and assayed for PDE modulating activity following resuspension in 10 mM formic acid.
PAC Purification of the MessengersThe protocol for this procedure is outlined in Table I, below.
PAC Purification of PDE-messengers1. Extraction of supernatant with chloroform/methanol
2. DE-52 anion exchange
3. C-18 reversed phase
4. Dowex 50W X 8 cation exchange
5. Charcoal adsorption
6. QAE-Sephadex anion exchange
7. aSAX HPLC anion exchange
8. P-2 gel filtration
Following extraction from the supernatants of liver membranes, PDE modulating activity was chromatographed on a 40 ml column of DEAE-Cellulose (DE-52), washed with 2 column volumes of 0.05M ammonium formate, pH 4.0 and eluted with 2 column volumes of 0.25M TEA-formate, pH 3.75. This fraction was chromatographed on 30 ml of C-18 reversed phase resin, loaded and eluted in 0.25M TEA-formate, pH 3.75.
PDE modulating activity was not retained on this column. Following lyophilization, sample was resuspended in 20 ml of 0.05M TEA-formate, pH 3.0, and chromatographed on a 20 ml Dowex 50W X-4 cation exchange column, eluted in the same buffer. Under these conditions, activity was not retained on this column. The eluate was lyophilized, resuspended in 0.05M TEA-formate, pH 4.5 and treated with activated charcoal for 10 min at 4°C The nonadsorbed supernatant was then chromatographed on a 10 ml QAE-Sephadex column, eluted with a linear gradient of 0.05M TEA-formate, pH 4.75 (FIG. 1). One ml fractions were lyophilized, resuspended in 10 mM formic acid and assayed for PDE modulating activity.
Two species of activity were detected, eluting at approximately 0.25 (I) and 0.45M (II) TEA-formate. Each peak was then chromatographed on an analytical SAX HPLC column (FIG. 2). One ml fractions were lyophilized and assayed. Peaks I and II eluted as distinct components of activity at 15 and 18 min respectively. The active fractions were then separately chromatographed on a 25 ml P-2 gel filtration column (FIG. 3). Under these conditions both peaks eluted with the same approximate volume as did bacitracin (MW =1400).
PDE Modulating activities were further characterized by high voltage thin layer electrophoresis (FIG. 4). Both substances were electrophoresed at pH 3.5 and 1.9, eluted from plates and assayed. Each species of activity migrated towards the anode although Peak II migrated slightly farther than Peak I at pH 3.5. At pH 1.9, Peak I migrated in a broader region, indicating the possible conversion of some fraction of this activity.
In crude extracts of plasma membrane derived supernatants, production of pyruvate dehydrogenase modulating activity was enhanced by insulin. These results were confirmed for production of PDE modulating activities (Table II, below).
| TABLE II |
| ______________________________________ |
| Effect of Insulin and Phospholipase C on Generation of PDE |
| Messenger from Liver Membranes |
| PDE Activity |
| Membrane Treatment |
| pmol mg protein-1 min-1 |
| (% Basal) |
| ______________________________________ |
| none 78.5 (141) |
| 1.0 nM Insulin |
| 138.0 (246) |
| 1.0 g/ml PI-PLC |
| 191.3 (341) |
| Insulin + PI-PLC |
| 197.5 (352) |
| ______________________________________ |
In the tests the results of which are tabulated above, liver membranes were treated as indicated for 10 min at 37°C, centrifuged and supernatants were extracted and purified up to and including the Dowex 50WX-4 step as described earlier herein under "Experimental Frocedures".
Partially purified extracts were assayed for ability to modulate cAMP phosphodiesterase. Basal activity was 56.2 pmoles/mg protein-1 -min-1.
Insulin treatment of plasma membranes resulted in a 4-fold stimulation of PDE modulating activity detected after partial purification. To probe the mechanism by which this process occurs, purified PI-PLC from S. aureus was added to membranes at a final concentration of 1.0 ug/ml. This enzyme is known to hydrolyze PI and release proteins which are covalently linked to phospholipids via an inositol containing glycan anchor. The enzyme is free of protease activity and does not hydrolyze polyphosphoinositides or other phospholipids. (Further purification of the PI-PLC is described in Example III).
PI-PLC addition caused a 6-fold increase in the production of messengers from membranes (Table II). Moreover, pretreatment of membranes with PI-PLC for 30 min at 37°C, followed by washing, prevented the further release of activity by insulin (data not shown), suggesting that prolonged exposure of plasma membranes to exogenously added PLC depleted the substrate for the action of insulin. Further purification of the messengers produced by PI-PLC revealed two peaks of activity with chromatographic and electrophoretic properties identical to those produced by insulin (data not shown).
It was attempted to identify the membrane associated substrate for PI-PLC, which serves as a precursor for generation of the messengers. Organic extracts of hepatic membranes were chromatographed on TLC as described in "Experimental Procedures" and lipids were eluted, treated with PI-PLC and extracted (FIG. 5). A single spot of precursor activity was detected on TLC (Rf=0.28), which migrated faster than phosphatidylinositol-4' phosphate, but slower than PI. Further purification of the PI-PLC generated activity revealed two PDE modulating substances with chromatographic properties identical to those produced from membranes by insulin (data not shown).
The dose dependence of action of Peaks I and II were evaluated (FIG. 6). The response of cAMP PDE to both substances was biphasic with respect to concentration, suggesting that the previously described separation of antagonistic regulators may have reflected resolution of two distinct compounds, each of which similarly exhibited paradoxical dose responses yet were present in dissimilar concentrations or with dissimilar potencies.
Several chemical properties of the biological activities residing in Peaks I and II were evaluated, as summarized in Table III, below.
| TABLE III |
| ______________________________________ |
| Chemical Modification of the Messengers |
| PDE Stimulation |
| Treatment (% Control) |
| ______________________________________ |
| none 100 |
| 0.1% TFA, 3 hrs. 100 |
| 10% TFA, 3 hrs. 10 |
| 0.1% NH4 OH, 3 hrs. |
| 21 |
| 10% HN4 OH, 3 hrs. |
| 0 |
| Diazomethane, 1 hr. 0 |
| Methanolic HCl, 1 hr. |
| 0 |
| Na Periodate: .01 M, 2 hrs., pH 6.0 |
| 100 |
| Na Periodate: 0.1 M 24 hrs., pH 6.0 |
| 27 |
| Na Nitrite: 0.2 M, 3 hrs., pH 3.75 |
| 29 |
| ______________________________________ |
With regard to the above data, purified PDE modulating activity residing in Peak I was subject to indicated treatments at room temperature, and assayed for ability to stimulate PDE. Results are expressed as % stimulation of activity with untreated mediator. For diazomethane treatment, control activity was measured after exposure to diethyl ether for 1 hr, which had no effect on activity. For methanolic HCl treatment, control activity was measured in 100% methanol, which did not affect activity. Periodate and nitrite treatments were compared to treatments in buffer alone, which did not affect activity. Identical results were obtained for activity residing in Peak II.
Activities of both substances exhibited similar susceptibility to strong acid and base. Each was inactivated by methylation with diazomethane or methanolic HCl. Each was partially susceptible to periodate oxidation, perhaps suggesting a carbohydrate structure without readily oxidizable hydroxyl groups. Deamination of glycosidically linked glucosamine with nitrous acid at pH 3.75 simultaneously converts the residue to the 2,5 anhydromannose derivative with hydrolysis of the glycosidic linkage. PDE modulating activities residing in both peaks were 80% inactivated by nitrous acid deamination at pH 3.75.
The above experiment details the purification of two substances from hepatic membranes which modulate the activity of the low Km cAMP phosphodiesterase in fat cells. These substances similarly regulate pyruvate dehydrogenase, adenylate cyclase and acetyl CoA carboxylase. They are rapidly produced in response to physiological levels of insulin, suggesting a role as mediators of some of the action of the hormone.
Several properties of these messenger materials have been studied. The acid stability, alkaline lability and non-nucleotide nature of these activities have been previously suggested, as well as a molecular weight of 1000-2000 daltons. Although a peptide structure has been suggested the activities of the purified substances described here were unaffected by proteolytic enzymes. Moreover, neither substance reacted with ninhydrin (data not shown). Behavior on ion exchange columns and migration on high voltage electrophoresis suggest that the two components contain dissimilar net negative charges, even at pH 1.9. This indicates the presence of phosphate or sulfate as a charged functional group. Both substances appear relatively polar, based upon water solubility and inability to absorb to reversed phase resin. Limited oxidation by periodate indicated the presence of carbohydrate in the active substance. This was further supported by loss of activity after nitrous acid deamination, which implied glycosidically linked glucosamine in the active compound. The presence of an inositol phosphate derivative was strongly suggested by the generation of the activities by a PI specific PLC. Preliminary carbohydrate analysis of the purified fractions after methenolysis revealed inositol phosphate, glucosamine and other monosaccharides.
As has been described, two species of messenger activity can be detected. Although previously suggested to produce antagonistic regulation of insulin target enzymes, it is now believed that these substances share similar bimodal activities with respect to dose dependence. Dissimilar concentrations of substance which both produce paradoxical dose responses, perhaps with different potencies, may explain the apparent resolution of stimulatory and inhibitory activities. Biphasic responses of target enzymes to these substances may account for the paradoxical effects of insulin on lipolysis, adenylate cyclase, and cAMP PDE. Aside from apparent differences in charge and solubility, no difference in the size or chemical sensitivities of these two substances were detected. Since they are both generated by PI-PLC hydrolysis of intact membranes or of an isolated putative glycolipid precursor, one possibility is that they may represent a 1,2-cyclic phosphate and 1- or 2-monophosphate inositol derivative, as has been described for the phosphodiesteratic cleavage of the phosphoinositides and the PI anchored variant surface glycoprotein from Trypanosoma brucei.
Of considerable interest was the finding that the substrate for the PI-PLC catalyzed generation of the messenger materials could be identified by the organic extraction of liver membranes. The identification of this putative precursor as an inositol and glucosamine containing glycolipid has been verified in primary cultures of hepatocytes and cultured myocytes by preincubation of cells with [3 H]inositol and [3 H]glucosamine. In these experiments which follow below, radioactivity from both sources was incorporated into a glycolipid with a Rf on TLC identical to the precursor described here. The radioactivity in this precursor was rapidly diminished by insulin in intact cells and by treatment of extracted lipids with exogenous PI-PLC. [3 H]inositol and [3 H]glucosamine were rapidly incorporated into fractions containing purified messenger in response to both insulin (cells) and PI-PLC (extracted lipids).
The studies on enzymatic production of the activities of the messenger materials purified and characterized in Example I with a PI-specific PLC, and evaluation of chemical sensitivities suggested that the enzyme modulating activity of these messengers was derived from substances which contained inositol and glycosidically linked glucosamine. In the present example, the actions of insulin of the production of the messenger materials were evaluated in the cultured murine myocyte line BC3 Hl. These cells become responsive to insulin upon differentiation following attainment of confluence. The insulin sensitive incorporation of radioactive inositol and glucosamine into HPLC fractions which contain activities ascribed to the messengers was observed. The production of these radioactive products mirrors that of the enzyme modulating activities, and appears to be due to the hydrolysis of a novel glycolipid precursor.
The purification procedure of Example I was modified for purification of similar substances from BC3 Hl cells incubated with [3 H]inositol or [3 H]glucosamine. Following insulin treatment and extraction, the aqueous phase was sequentially chromatographed on DEAE-Cellulose, C-18 reversed phase and Dowex 50WX-4 cation exchange. This was followed by chromatography on an SAX HPLC column (FIG. 7). Insulin treatment increased the incorporation of [3 H]inositol into two peaks which eluted at 15 (I) and 18 (II) min. Minimal incorporation into these fractions was observed in control cells (FIG. 7a). Insulin had a similar effect on incorporation of [3 H]glucosamine into identical fractions (FIG. 7b), although another radioactive peak was detected at 23 min. which was unaffected by insulin. These two peaks of insulin stimulated radioactivity exhibited distinct retention times from those of 1,2 cyclic inositol monophosphate (10 min.) or inositol monophosphate (24 min.). However, Peaks I and II coeluted with two peaks of PDE modulating activity produced in these cells in response to insulin. These myocyte derived activities were indistinguishable from those purified from insulin treated liver plasma membranes.
PDE messengers can be produced from liver membranes by addition of PI-PLC from S. aureus. These enzyme specifically hydrolyzes the phosphodiester linkage of phosphatidylinositol and those proteins covalently attached to PI via a glycan anchor. These preparations of PI-PLC were free of protease activity, and were inactive against polyphosphoinositides and other phospholipids. Cells were preincubated with [3 H]inositol or [3 H]glucosamine, and lipids were extracted and treated with PI-PLC. After a 30 min. incubation, cells were extracted, the aqueous products were purified as described above and chromatographed on an SAX HPLC column (FIG. 8). PI-PLC treatment resulted in the generation of two peaks containing [3 H]inositol which exhibited retention times identical to those produced by insulin in intact cells. [3 H]glucosamine was also incorporated into these fractions in response to PI-PLC, although a spontaneously released peak was observed at 23 min. which was unaffected by PI-PLC.
Described above is a chemically unidentified glycolipid in liver plasma membranes which appeared to serve as a substrate for the generation of PDE messengers by the S. aureus PI-PLC. The incorporation of [3 H]inositol and [3 H]glucosamine into this putative precursor was examined in BC3 Hl cells. Thin layer chromatography of extracted phospholipids revealed a peak into which both [3 H]inositol and [3 H]glucosamine were incorporated (FIG. 9). This glycolipid exhibited an Rf identical to the putative precursor of the PDE messenger from liver. It migrated faster than phosphatidylinositol-4-phosphate but slower than phosphatidylinositol. After treatment of cells with insulin for 10 min., the incorporation of both [3 H]inositol (FIG. 9a) and [3 H]glucosamine (FIG. 9b) in this peak was increased approximately 40%, with no significant change in labeling of other lipids. In parallel experiments, lipids were extracted from labeled cells and treated with or without PI-PLC for 30 min. (FIG. 10). Exposure to this enzyme caused a 60% decreased in [3 H] inositol labeling (FIG. 10a) and 40% decrease in [3 H]glucosamine labeling (FIG. 10b) of an identical spot on PLC. As expected, PI was 50% depleted by the S. aureus PI-PLC. Similar results, have been observed by PI-PLC treatment of plasma membranes prepared from these labeled myocytes.
The time course of radioisotopic incorporation into the products and putative precursor described above was examined after exposure to cells to 10 nM insulin (FIG. 11). In these experiments, the water soluble products were sequentially chromatographed as described above, and results represent a combination of Peaks I and II from HPLC. Insulin rapidly stimulated the production of these [3 H]inositol containing products. Seventy percent of the maximal effect was achieved after 30 sec. The time course of [3 H]inositol incorporation into the precursor (as identified on TLC in FIGS. 9 and 10) was biphasic. Insulin caused a small (20%) but significant decrease in counts at 30 sec. Thereafter, radioactivity in this peak increased, resulting in a 60% increase over control levels.
The phosphodiesteratic cleavage of a phospholipid by insulin should also be reflected by increased production of diacylglycerol, which could serve as substrate for a diglyceride kinase, producing phosphatidic acid. Myocytes were preincubated with [3 H]myristic acid and both diacylglycerol and phosphatidic acid labeling were evaluated by thin layer chromatography (FIG. 6). Insulin caused a 40% increase in [3 H]myristate diacylglycerol within 1 minute. Labeled diacylglycerol decline to near basal levels by 2 minutes, and then increased, reaching a maximal 50% increase over basal by 5-10 minutes. Labeled diacylglycerol production was followed by a 2 fold increase in levels of [3 H]myristate phosphatidic acid, achieved by 5 minutes. [3 H]myristate diacylglycerol was also rapidly produced by incubation of extracted lipids with S. aureus PI-PLC (data not shown).
The chromatographic properties of the [3 H]inositol and [3 H]glucosamine labeled products from insulin treated BC3 Hl cells were further evaluated. Pooled radioactivity residing in SAX Peak I was chromatographed on a P-2 gel filtration column (FIG. 13). The elution profiles of [3 H]inositol (FIG. 13a) and [3 H]glucosamine (FIG. 13b) labeled products were compared with that of the similarly purified myocyte derived PDE modulating activity (FIG. 7c). Each substance eluted in an identical volume indicating an approximate molecular weight of 1400. Both radioactive and bioactive substances in Peak II exhibited an identical elution on P-2 (data not shown). Following P-2 gel filtration, active samples were pooled and subject to high voltage electrophoresis on cellulose coated thin layer plates (FIG. 14). The electrophoretic migration of both labeled products in Peak I was identical to that of the PDE messenger. Similar results were obtained when samples were electrophoresed at pH 1.9. Radioactively labeled products and PDE messenger activity residing in Peaks II migrated slightly farther than that of Peak I at both pHs (not shown) as described previously, although comigration of radioactivity and bioactivity was again observed. These results confirm previous findings of dissimilar net negative charge for these compounds, implicating phosphate or sulfate as charged species.
To further evaluate differences in the two products each of the [3 H]inositol labeled peaks was digested at 25°C for 16 hrs. with alkaline phosphatase (100 U/ml) in 50 mM ammonium bicarbonate, pH 8∅ Each sample was then injected injected onto a SAX column, and counts in each peak were determined. Alkaline phosphatase treatment had no effect on counts of Peak I, but produced a 70% loss of counts in Peak II as shown in Table IV, below.
| TABLE IV |
| ______________________________________ |
| Modification of [3 H] Inositol Labeled Products |
| dpm |
| Treatment Peak I Peak II |
| ______________________________________ |
| none 1820 1100 |
| Alkaline Phosphatase |
| 1963 341 |
| (10 g/ml) |
| Nitrous Acid 220 195 |
| (0.2 M, pH 3.75) |
| ______________________________________ |
Following gel filtration, purified [3 H]inositol labeled products in Peaks I and II were treated with (a) alkaline phosphatase (10 U/ml) in 50 mM ammonium bicarbonate, pH 8.0 for 16 hrs. at 30°C , or (b) 0.2M Na Nitrite in 50 mM ammonium acetate, pH 3.75 for 3 hrs. at 25°C Following incubation, samples were injected into SAX HPLC column, eluted as detailed in FIG. 7, and collected in buffer alone, all counts were recovered with the appropriate retention time.
These results were consistent with the possibility that Peak I contains a cyclic 1,2 phosphodiester inositol derivative which is not susceptible to alkaline phosphatase, whereas Peak II may contain phosphomonoester inositol derivative which is hydrolyzed by alkaline phosphatase.
Treatment with nitrous acid at pH 3.75 deaminates glycosidically linked glucosamine, converting the residue to a 2,5 anhydromannose derivative involving cleavage of the glycosidic linkage. Treatment of purified [3 H]inositol labeled products in both peaks with nitrous acid caused an 80% loss of counts detected after HPLC (Table IV). These results were verified by measurement of radioactivity on high voltage electrophoresis. Eighty-five percent of [3 H]inositol migrated as inositol monophosphate after nitrous acid deamination (data not shown).
Upon differentiation, the cultured myocyte line, BC3 Hl, develops affinity insulin receptors and several biological responses to the hormone, including glucose transport, amino acid uptake, insulin receptor down-regulation and increased labeling of certain phospholipids. The above experiments confirm that the apparent phosphodiesteratic cleavage of a chemically unidentified glycolipid resulting in the generation of two water soluble products as well as diacylglycerol. Both the products and putative precursor of the reaction have been identified by radioisotopic labeling with [3 H]inositol and [3 H]glucosamine. These labeled compounds appear to be incorporated into the water soluble products with a 1:1 stoichiometry. The insulin dependent hydrolysis of the putative glycolipid precursor, and subsequent appearance of the products occurs in less than 1 min. Thereafter, [3 H]inositol incorporation into the precursor increases, suggesting its rapid resynthesis. This process appears similar to that observed in the hormone stimulated phosphodiesteratic cleavage of the phosphoinositides, in which these phospholipids are rapidly replenished.
The hydrolysis of the putative precursor described here and subsequent generation of carbohydrate products and diacyglycerol were also accomplished by treatment of extracted phospholipids with S. aureus PI-PLC, which specificially hydrolyzes phosphatidylinositol and releases proteins covalently anchored to phosphatidylinositol. Thus, insulin action may involve activation of an endogenous PLC activity which hydrolyzes a novel inositol/glucosamine containing glycolipid. Since insulin does not stimulate hydrolysis of polyphosphoinositides, the insulin sensitive PLC may be similar to the PLC activity identified in Trypanosoma brucei which selectively attacks the glucosamine/inositol phosphodiester anchor of the membrane bound variant surface such a selective PLC in insulin action is supported by observations that the purified trypanasoma PLC is an effective as the S. aureus enzyme in hydrolysis of the myocyte glycolipid precursor.
The water soluble products of the putative phosphodiesteratic hydrolysis stimulated by insulin have been purified and characterized. Coincorporation of [3 H]inositol and [3 H]glucosamine was observed into identical fractions on distinct chromatographic and electrophoretic systems. Characterization of net charge (ion exchange), size (gel permeation), mass charge ratio (high voltage electrophoresis) and hydrophobicity (reversed phase) indicated that labeled inositol and glucosamine are incorporated into the same products in an insulin dependent manner. Although it is possible that these labeled precursors were first converted to other sugars during preincubation, the incorporation of intact myo-inositol was supported by the selective hydrolysis of the putative precursor and generation of product by PI-specific phospholipase C. Moreover, glucosamine incorporation was validated by loss of radioactivity in purified fractions after nitrous acid deamination under conditions specific for cleavage of glycosidically linked glucosamine.
The insulin sensitive products labeled with [3 H]sugars cochromatographed with insulin stimulated messengers, derived both from BC3 Hl myocytes and liver plasma membranes. Bioactivity and radioactivity from those sources exhibited identical electrophoretic and chromatographic behavior on each system and under all conditions. PDE modulating activities were produced by insulin over the same time course and concentration range as were the purified radiolabeled products (data not shown). Identical species of bioactivity and labeled products were generated by treatment of membranes or extracted phospholipids with S. aureus PI-PLC. These results indicated that the activities of the insulin generated PDE messengers can be ascribed to the radiolabeled complex carbohydrates described here.
The two insulin sensitive, radiolabeled products from myocytes resolved by ion exchange chromatography to correlate with the two species of PDE modulating activity derived from liver. These two peaks share several chemical properties, yet exhibit dissimilar net negative charges, even at pH 1.9. Since both substances were generated by PI-PLC hyrolysis of a membrane glycolipid, one possibility is that they represent a 1,2-cyclic monophosphate (Peak I) and 1- or 2-monophosphate (Peak II) inositol derivatives. Such cyclic products have been identified for the phosphodiesteratic cleavage of phosphoinositides and the PI anchored variant surface glycoprotein from Trypanosoma brucei. This hypothesis is supported by the susceptibility of [3 H]inositol labeled Peak I was unaffected by the enzyme.
The chemical identity of the putative glycolipid which serves as substrate for insulin stimulated hydrolysis remains undefined. This novel glycolipid shares some characteristics with the membrane glycan anchor described fro the trypansoma variant surface glycoprotein, which consists of phosphate, glucosamine and myo-inositol as well as ethanolamine and other carbohydrates. It appears that insulin causes the phosphodiesteratic cleavage of such a phospholipid, resulting in the generation of two closely related carbohydrate products which may mediate some of the acute actions of insulin. Included in this process is the obligatory production of diacylglycerol, the endogenous activator of protein kinase C. Although diacylglycerol production by insulin was previously attributed to stimulation of de novo synthesis,the transient generation of diacylglycerol has been observed in response to insulin, followed by the apparent rapid phosphorylation to phosphatidic acid. Diacylglycerol and the [3 H]inositol labeled products appeared to arise from the same precursor, and their production was kinetically indistinguishable. It is possible that this unique pathway of diacylglycerol production may provide a selective mechanism for insulin's regulation of protein kinase C activity, and perhaps explain why phorbol esters reproduce some but not all of the effects of insulin. The further characterization of the molecular components involved in this process, as well as the chemical identities of the aqueous products, may elucidate many of the molecular mechanisms of insulin action.
Among the activities previously detected in the crude extracts of insulin treated cells or plasma membranes were modulation of pyruvate dehydrogenase (PDH) and adenylate cyclase. In the study that follows, the regulation of these enzymes by the present messenger compounds is characterized. These results indicate that the inositol-glycan enzyme modulator may be in part responsible for the regulation by insulin of cAMP levels and fatty acid synthesis.
PAC MaterialsMost reagents were from Sigma, with the exception of collagenase (Millipore), DEAE-Cellulose and SAX HPLC columns (Whatman), P-2 gel filtration media, Dowex AG-LX8 and 50 W X-4 (Bio-Rad), pyruvate kinase (Boehringer Mannheim) bovine insulin (Burroughs Wellcome), QAE-Sephadex (Pharmacia), 2,8[3 H]cAMP and [32P]ATP (NEN) and [14 C]pyruvic acid (Amersham). Male rats (100-125 g) were from Sprague-Dawley. Purified PI-PLC from S. aureus was a generous gift of Dr. Martin Low of the Oklahoma Medical Research Foundation. This enzyme is free of protease activity and was inactive in the hydrolysis of polyphosphoinositides and other phopholipids [Low, M. G. Zilversmit, D. B., (1980) "Role of Phosphatidylinositol in Attachment of Alkaline Phosphatase to Membranes", BIOCHEMISTRY, 19:3913].
Purification of the Enzyme Modulator. Bovine liver particulate fraction was prepared as described previously [Saltiel A. R., (1984) "Preparation and Characterization of Putative Insulin Mediators from Liver", METHODS IN DIABETES RESEARCH, eds. J. Larner, S. Pohl, I:73]. Solutions were incubated in 5 mM ammonium bicarbonate, pH 7.4 with 10 nM insulin or 1 ug/ml PI-PLC as described in the figure legends. Following incubation, solutions were acidified to pH 4.0 and centrifuged at 35,000 x g for 20 minutes. Resulting supernatants were extracted with chloroform/methanol (2:1). The upper aqueous phase was aspirated and methanol was evaporated. Enzyme modulating activities were purified as described in Publication No. 6 listed earlier herein. Briefly, the extract was chromatographed on DEAE-Cellulose and eluted with 0.25M triethylamine (TEA)-formate, pH 3.75. This was followed by chromatography on C-18 reversed phase resin, in the same buffer, followed by Dowex 50W X-4 in 50 mM TEA-formate, pH 3∅ The resulting eluate was lyophilized, resuspended in 50 mM TEA-formate, pH 4.5 and sequentially chromatographed on QAE-Sephadex, SAX HPLC and a P-2 gel filtration column. Only the cyclic inositol phosphate derivative residing in Peak I was examined.
Enzyme Assays. The low Km cAMP PDE was assayed in a rat adipocyte particulate fraction as previously described [Saltiel, A. R., Steigerwalt, R. W., (1986) "Purification of Putative Insulin-Sensitive cAMP Phosphodiesterase or its Catalytic Domain From Rat Adipocytes", DIABETES, 35:698] and as later modified in Publication No. 6. Activity was linear throughout 20 min. and proportional to protein concentration. Only the low Km species of the enzyme was present in this fraction. PDH activity was assayed in liver mitoplasts after preincubation with ATP as previously described [Saltiel et al., (1982) PROC. NATL. ACAD. SCI. U.S.A., 79:3513 Adenylate cyclase activity was also assayed in purified adipocyte plasma membranes. For all enzyme assays, variability was less than 5%.
PAC Insulin and PI-PLC Stimulate the Generation of an Enzyme ModulatorPlasma membranes were treated with 10 nM insulin or 1 ug/ml PI-PLC for 10 min. and the resulting supernatants were partially purified and assayed for enzyme modulating activity. The results of the assays are set forth in Table V appearing later on herein. As observed previously, some enzyme-modulating activity was spontaneously released from control membranes. Exposure of membranes to insulin caused the generation of a substance which produced a 2-3 fold stimulation of PDE and PDH and a 75% inhibition of adenylate cyclase. Incubation of liver membranes with S. aureus PI-PLC also caused the equivalent production of a stimulator of PDE and PDH and an inhibitor of cyclase. Preincubation of the liver membranes with PI-PLC, followed by washing, prevented the further generation of enzyme modulators by insulin (data not shown). Further purification of the insulin or PI-PLC generated activities revealed no chromatographic differences.
Supernatants from insulin treated plasma membranes were extracted and purified up to and including the P-2 gel filtration column. The eluted fractions from this column were assayed for ability to modulate PDE, PDH or adenylate cyclase. Stimulation of PDE and PDH and inhibition of cyclase activities were detected in identical fractions corresponding to an approximate molecular weight of 1400. Some of the chemical properties of the purified substance were also examined by evaluating ability to modulate the activities of the enzymes after exposure to several reagents. This data is set forth in Table VI appearing below.
None of the enzyme-modulating activities were altered by digestion with trypsin, chymotrypsin, papain or pronase (not shown). Each enzyme-modulating activity was destroyed by hydrolysis in strong acid at 110°C although each was stable to mildly acidic conditions. In contrast, the three enzyme-modulating activities were all alkaline sensitive. Methylation with methanolic HCl destroyed each enzyme activity. Twenty-four hr. incubation with sodium periodate resulted in 70-80% loss of activity. Exposure to nitrous acid at pH 3.75 caused 70-85% inactivation of each enzyme-modulating activity, suggesting deamination of glucosidically linked glucosamine in the active compound.
The effect of the purified enzyme modulator on adenylate cyclase activity was evaluated and the results set forth in Table VII. Cyclase activity was diminished by 70-80% under basal, forskolin-, Gpp(NH)p- and isoproterenol-stimulated conditions. Fluoride-stimulated activity was inhibited only 30% with the same dose of modulator. The kinetics of action of the modulator were also examined. Evaluation of the dependence of the enzyme on substrate concentration revealed that the modulator diminished the Vmax of the enzyme with no apparent effect on the Km. The inhibition of the enzyme was also evaluated as a function of metal ion concentration. Although the enzyme modulator caused inhibition of cyclase activity at all concentrations of MgCl2 tested, the effect on the MgCl2 response was more complex. The addition of MgCl2 to fat cell cyclase produced a biphasic response with respect to concentration which has been previously observed in other cyclase system, and may reflect Mn++induced uncoupling of cyclase from its regulatory protein(s). The modulator caused an inhibition of enzyme activity at MnCl2 levels (less than 0.5 mM) which were stimulatory, but produced less inhibition at higher concentrations of this divalent cation.
The regulation of rat liver PDH by the purified enzyme modulator was evaluated. As was described previously for the regulation of this enzyme by crude extracts from insulin treated cells or plasma membranes, stimulation of PDH activity was rapid, dose dependent and could be blocked by 100 mM NaF, suggesting activation of the PDH phosphatase as a mechanism of action. The effect of the enzyme modulator on the substrate dependence of PDH activity was evaluated. The modulator caused an increase in the Vmax of the enzyme, with no apparent effect on the Km. The dependence of PDH activity on pH was also evaluated in the presence or absence of the purified modulator. Stimulation of enzyme activity was optimal at pH 6.5-7.5, but declined at pH 8∅
| TABLE V |
| ______________________________________ |
| GENERATION OF AN ENZYME MODULATOR FROM |
| LIVER PLASMA MEMBRANES |
| CON- INSULIN- PI-PLC |
| NO TROL TREATED TREATED |
| ADDI- MEM- MEM- MEM- |
| TION BRANES BRANES BRANES |
| ______________________________________ |
| cAMP 61.7±4.2 |
| 78.5±5.6 |
| 146±3 192±5 |
| Phospho- (311.1) |
| diesterase1 |
| Pyruvate |
| 1.77±0.07 |
| 2.45±0.20 |
| 4.80±0.38 |
| 5.25±0.40 |
| Dehydro- (296.6) |
| genase2 |
| Adenylate |
| 272±14 |
| 210±4 72.0±3.7 |
| 68.7±7.6 |
| Cyclase1 (25.0) |
| ______________________________________ |
Liver membranes were treated with or without 10 nM insulin or 1 ug/ml PI-PLC for 10 min. at 37°C Following cetrifugation, the supernatants were extracted and purified up to and including the SAX HPLC step. Purified extracts were added to enzyme assays at a final dilution of 1:10. Enzyme activities were assayed with no additions or after addition of purified extracts from control, insulin or PIPPLC treated membrane. Percent of basal activity is expressed in parentheses. 1 expressed as pmoles/mg protein-min. 2 expressed as nmoles/mg protein-min.
| TABLE VI |
| ______________________________________ |
| EFFECT OF THE ENZYME MODULATOR ON |
| ADENYLATE CYCLASE ACTIVITY |
| 10 uM + |
| 10 uM 10 uM 100 mM 0.1 mg/ml |
| BASAL Gpp (NH) p |
| iPT NaF Forskolin |
| ______________________________________ |
| Control |
| 310 ± 12 |
| 241 ± 10 |
| 702 ± 31 |
| 869 ± 57 |
| 657 ± 52 |
| Enzyme 85.1 ± 7 |
| 65.4 ± 5 |
| 197 ± 12 |
| 594 ± 31 |
| 208 ± 17 |
| Modu- |
| lator |
| ______________________________________ |
Adenylate cyclase activity was assayed in fat cell plasma membranes in the presence of the indicated additives, with or without the purified enzyme modulator. Results are expressed as pmoles cAMP mg protein-1 min-1.
| TABLE VII |
| ______________________________________ |
| CHEMICAL MODIFICATION OF THE |
| ENZYME MODULATOR |
| Enzyme-Modulating Activity (% control) |
| Treatment PDE PDH ADENYLATE CYCLASE |
| ______________________________________ |
| Control 100 100 100 |
| 10% NH4 OH, 100°C |
| 0 0 0 |
| 0.1% TFA, 100°C |
| 87 100 122 |
| 10% TFA, 100°C |
| 24 32 38 |
| Methanolic HCl |
| 0 5 17 |
| Na Periodate, 24 hrs. |
| 25 30 19 |
| Na Nitrite, 3 hrs. |
| 21 15 31 |
| ______________________________________ |
The purified enzyme modulator was exposed to the indicated treatments, and assayed for the ability to stimulate PDE or PDH or inhibit adenylate cyclase. Results are expressed as % modulation of activity with untreated modulator. NH4 OH and TFA treatments were carried out for 2 hrs. Methanolic HCl was added for 0.5 hrs. at 25°C Control activity was measured by resuspension in 100% methanol, which did not affect activity. Na Periodate and Na Nitrite were added at a final concentration of 0.2M in 50 mM ammonium acetate buffer, pH 6.0 (periodate) or pH 3.5 (nitrite) at 37°C These were compared to treatment in buffer alone, which did not alter activities.
In this study, the precursor of the invention has been isolated and further characterized. This work commenced from the proposition that insulin-stimulated diacylglycerol production in BC3 Hl cells is due to the action of a phospholipase C which selectively cleaves a PI-glycan substrate, generating both diacylglycerol and the inositol-glycan which modulated certain insulin sensitive enzymes. Moreover, the selective action of this insulin sensitive PLC on the PI-glycan may result in the exclusive gneration of myristate-labeled diacylglycerol, which can be distinguished from the arachidonate containing diacylglycerol derived from the hormone-stimulated hydrolysis of the phosphoinositides in these cells.
PAC MaterialsAll reagents were from Sigma, with the exception of tissue culture medium (Flow Laboratories), Nu Serum (Collaborative Research), Collagenase (Millipore), Dowex AG-IX8 (BioRad), bovine insulin (Burroughs Wellcome), 2,8[3 H]cAMP, [2,63 H]inositol, [1,63 H]glucosamine, [9,103 H]myristic acid and [5,6,8,9,11,12,14,153 H]arachidonic acid (New England Nuclear). The analytical SAX HPLC column was from Whatman, LC Silica HPLC column was from Supelco and silica gel G plates were from Fisher. Male rats (100-125 g) were from Sprague-Dawley. Purified PI-PLC was the generous gift of Dr. Martin Low of the Oklahoma Medical Research Foundation. This enzyme was free of protease activity, and was inactive in the hydrolysis of polyphosphoinositides and other phospholipids.
BC3 Hl cells were cultured on collagen coated multiwell plates or petri dishes in Dulbecco's minimal essential medium (DMEM) supplemented with 20% Nu serum or 10% fetal calf serum. Cells were grown to maximal density and confluence, at which time they become responsive to insulin. Radiolabeled precursors were added for 20 hrs. in serum-free medium. [3 H]Myristic and arachidonic acids were complexed 1:1 to bovine serum albumin prior to addition.
Following preincubations with the appropriate isotopes, cells were resuspended in DMEM without serum and treated with 10 nM insulin at the designated intervals. Reactions were terminated by the addition of 1 ml chloroform/methanol/1N HCl (200:100:1), followed by 0.5 ml 10 mM formic acid. Following centrifugation at 500 x g for 5 min., the upper phase was discarded. For phospholipid analyses, the lower, organic phase was dried under N2, resuspended in chloroform/methanol/H2 O (9:7:2) and spotted on oxalate impregnated silica gel G plates. These were sequentially developed in chloroform/acetone/methanol/glacial acidic acid/H2 O (10:4:2:2:1) and chloroform/methanol/4N NH4 OH (45:35:10). One cm regions were scraped and eluted with chloroform/methanol (2:1) and counted. Phosphoinositide standards were identified by iodine staining.
For analysis of neutral lipids, the chloroform/methanol organic phases were dried under N2 and resuspended in 1 ml diethyl ether followed by addition of 1 ml 50 mM formic acid. The upper ether phase was aspirated and the lower aqueous phase was reextracted with 1 ml diethyl ether. The pooled ether phases were dried under N2, resuspended in chloroform and spotted on silica gel G plates which were preactiviated at 60°C for 1 hr. Plates were twice developed in petroleum ether/diethyl ether/glacial acetic acid (70:30:2). Five ug of unlabeled dimyristoyl glycerol, myristic acid and monomyristoyl glycerol were added as carriers to each sample.
The phospholipid precursor was further chromatographed on a silica HPLC column, eluted with a linear, 20 min. gradient of chloroform/methanol/glacial acetic acid/H2 O (65:25:10:1 to 40:45:10:5) at 1 ml/min. The aqueous products of the hydrolysis reactions were identified on an analytical SAX HPLC column, eluted with a linear 15 min. gradient of 60% methanol to 0.5M triethylamine-formate, pH 4.5 at 1 ml/min., as described previously by Saltiel, and Cuatrecasas, (1986), DIABETES, 35: 698-704.
PAC Diacylglycerol Production of BC3 Hl CellsThe time course of production of diacylglycerol was evaluated in response to hormones. Cells were labeled with [3 H]myristic acid or [3 H]arachidonic acid and exposed to insulin or epinephrine, known to act as an alpha-1 adrenergic agonist in these cells. In [3 H]myristate labeled cells, insulin caused a rapid increase in labeled diacylglycerol, which was maximal at 1 min., declined by 2 min. and slowly increased thereafter. Exposure to epinephrine resulted in no significant change in [3 H]myristoyl diacylglycerol above basal levels. In cells incubated with [3 H]arachidonic acid, insulin treatment caused a slow increase in labeled diacylglycerol. By contast, epinephrine produced a rapid increase in [3 H]arachidonyl diacylglycerol that was maximal at 1 min., declined at 5 min. and then slowly increased. Insulin had no detectable effect on the generation of [3 H]stearyl diacylglycerol.
Previously, a novel PI-glycan was identified which served as a precursor for the insulin-dependent generation of PDE-modulating substances in liver and muscle cells. Cultured BC3 Hl cells were separately incubated with [3 H]inositol, [3 H]glucosamine or [3 H]myristic acid for 20 hrs. Lipids were extracted, treated with or without PI-PLC purified from S. aureus, and chromatographed on thin layer plates. Extraction of phospholipids from inositol labeled cells revealed two tritiated spots which were diminished by incubation with PI-PLC. The PI-PLC sensitive spot migrating towards the solvent front coeluted with PI. The remaining PI-PLC sensitive spot, which migrated between phosphatidylinositol-4'phosphate and phosphatidylinositol, exhibited chromatographic behavior identical to the glycolipid identified as precursor for the PI-PLC generated PDE modulators. This spot was tentatively identified as the PI-glycan. Extraction of cells preincubated with [3 H]glucosamine revealed one major PI-PLC sensitive spot on TLC which comigrated with the PI-glycan. [3 H]Myristic acid was incorporated into several lipid species, although only the radioactivity residing in spots corresponding to PI and the PI-glycan were diminished by treatment with PI-PLC.
The radiolabeled PI-glycan could be purified further by an HPLC silica column. The region of the thin layer plate which contained the PI-glycan was scraped and eluted. The resulting substance was a poor substrate for hydrolysis by PI-PLC. However, when reconstituted by sonication with lipid vesicles containing 1:1 mixtures of phosphatidylethanolamine:phosphatidylcholine, complete hydrolysis was observed. The TLC-eluted lipid was reconstituted and incubated with or without PI-PLC for 2 hr., and then extracted with chloroform/methanol and phase separated. The organic phases were chromatographed on an HPLC silica column. A predominant peak containing labeled inositol or myristic acid was detected at 22 min. which was diminished in PI-PLC treated extracts. Twenty-four hr. incubation of this fraction with PI-PLC resulted in its complete hydrolysis.
After PI-PLC digestion of the PI-glycan, both the aqueous and organic products were analyzed. The labeled PI-glycan was purified by HPLC and treated with PI-PLC as described above. For glycolipid extracted from cells labeled with [3 H]inositol, the aqueous products were resolved by phase extraction and chromatographed directly on an HPLC SAX column. PI-PLC treatment caused the generation of one peak of radioactivity eluting at 15 min. This labeled substance eluted with a retention time distinct from that of cyclic 1,2 inositol monophosphate (9 min.) or 1- or 2-inositol monophosphate (24 min.). The PI-PLC generated radiolabeled substance corresponded to radiolabeled Peak I previously detected in insulin treated BC3 Hl cells. No radioactivity was detected in fractions corresponding to Peak II. Previous studies suggested that Peaks I and II contained cyclic and monophosphate inositol phosphate derivatives respectively. Detection of only Peak I by PI-PLC hydrolysis of the purified PI-glycan precursor followed by direct chromatography suggests that the cyclic compound may be the only product generated initially.
The identity of the labeled aqueous product as the previously detected, bioactive PDE-modulating substance was validated by an assay of the enzyme modulating activity produced from unlabeled precursor. Unlabeled cells were extracted and the PI-glycan was identified on HPLC by coelution with trace amounts of the labeled, purified glycolipid. The resulting silica HPLC purified PI-glycan was treated with or without PI-PLC, aqueous products were injected into an HPLC SAX column and fractions were assayed for PDE-modulating activity. A single PDE-modulating substance was identified with a retention time (15 min.) identical to that of the radiolabeled product. Further analysis of elution behavior on P-2 gel filtration columns and high voltage thin layer electrophoresis revealed that the radioactive and bioactive substances displaced identical chromatographic and electrophoretic properties.
To analyze the non-aqueous product of the PI-PLC catalyzed hydrolysis, the purified [3 H]myristic acid labeled PI-glycan was treated with or without PI-PLC, and subsequently extracted with ether. TLC of this neutral lipid fraction indicated that the PI-PLC treatment caused the generation of diacylglycerol which contained [3 H]myristic acid.
As set forth earlier, insulin stimulates the production of two related complex carbohydrate-phosphate substances in BC3 Hl cells containing inositol and glucosamine. The generation of these substances is accompanied by the production of diacylglycerol, followed by phosphatidic acid, and appear to result from the phosphodiesteratic cleavage of a novel PI-glycan. The putative insulin-sensitive PLC appears to catalyze the phosphodiesteratic cleavage of a novel glycolipid which contains PI, glucosamine and other monosaccharides and is labeled with myristic acid. This action of insulin causes the generation of two potential second messengers, (1) the inositol phosphate-glycan which modulates several insulin sensitve enzymes including cAMP phosphodiesterase, pyruvate dehydrogenase and adenylate cyclase, and (2) myristol diacylglycerol. Significantly, the insulin-stimulated hydrolysis of this glycolipid does not produce inositol trisphosphate, whereas epinephrine stimulates a phosphoinositide specific-PLC, resulting in the production of inositol phosphates and arachidonyl diacylglycerol. This proposed substrate specificity for hormonally regulated phosphodiesterase hydrolysis reactions is further supported by the recent identification of a PLC activity with a distinct substrate specificity for the PI-glycan and a related phosphoinositide glycoside. The study and data generated therefrom are set forth in Example V, below.
In this study, the enzyme, PI-glycan-specificphospholipase C (PLC), which catalyzes the phosphodiesteratic hydrolysis of insulin-sensitive PI-glycan precursor (precursor material) to produce soluble IP-glycans and diacylglycerol (DAG), is purified and further characterized. The location of the enzyme in the plasma membrane and its substrate specificity are consistent with its role as an effector in mediating some of the actions of insulin.
PAC MaterialsAll reagents were from Sigma, with the exceptions of Nonidet P-40 (NP-40) (Cal Biochem), DE-52 DEAE-cellulose (Whatman), Silica G and Silica 60 plates (Fisher), Extractigel D (Pierce), fetal calf serum (Gibco), defined minimal essential medium (Dulbecco's), bovine achilles tendon collagen (Worthington), and Silver-staining kit and Protein Assay (Bio-Rad). [9,10-3 H]phosphatidylcholine, Econofluor, and Aquasol were from New England Nuclear. The BC3 Hl myocyte cell line was the kind gift of Mary Standaert.
Preparation of Membrane Form VSG, C-terminal Glycopeptide, and Dimyristyl-phosphatidylinositol. 1010 trypanosomes of the Molteno Trypanozoan antigen type (MlTat) 1.4 purified from infected rat blood were biosynthetically labeled by incubation with [9,10-3 H]myristate. mfVSG was extracted from the labeled cells with 0.1% trifluoroacetic acid and purified by HPLC using a 5 um reversed-phase C-18 preparative Ultrapore column (Altex) as described previously in Fox et al., J. BIOL. CHEM., 261:15767-15771 (1986) and Clarke et al., MOL. BIOCHEM. PARASITOL., 17:19-34 (1985). Purified mfVSG was digested with pronase for 36 hr. to obtain the C-terminal glycopeptide. Dimyristyl-Pl was prepared from mfVSG by nitrous acid deamination. The identities of the two mfVSG derivatives were confirmed by thin layer chromatography (TLC).
Purification of PI-Glycan Precursor. Confluent cultures of BC3 Hl myocytes were serum-deprived and labeled with [3 H]inositol (0.5 uCi/ml medium) for 48 hr in serum free media. The labeled cells were extracted with chloroform:methanol:HCl (1N) (200:100:1) and the PI-glycan precursor was prepared by TLC, followed by HPLC as described in Example IV.
Protein assay and NaDodSO4 -PAGE. The protein concentration of PI-glycan PLC was determined by the method of Lowry et al. J. BIOL. CHEM., 193:264-275 (1951). after precipitation with TCA using prepackaged reagents from Sigma. mfVSG protein was assayed by the method of Bradford, ANAL. BIOCHEM, 72:248-254 (1976), with the Bio-Rad assay. NaDodSO4 -PAGE was performed using 10% polyacrylamide gels in Laemmli buffers . Proteins were visualized either by staining with Coomassie blue or silver.
Purification of Rat Liver Plasma Membranes. Plasma membranes were prepared from fresh livers obtained from 5-7, 350-500 g adult male Sprague-Dawley rats (Charles River Laboratories) by slight modification of the technique described by Neville, BIOCHEM. BIOPHYS. ACTA., 154:540-552 (1968). Briefly, livers were minced and homogenized in 1 mM sodium bicarbonate buffer that contained 10 mM EGTA, 5 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride, and 10 ug/ml aprotinin, leupeptin, pepstatin A, and chymostatin. All other buffers used during membrane purification contained 0.1 mM phenylmethylsulfonyl-fluoride. The liver homogenate was centrifuged for 10 minutes at 400 x g and the pellets were resuspended in 60% (w/v) sucrose to achieve a final sucrose concentration of 44%. Ten ml of 40% sucrose were layered over 25 ml of the homogenate, and the preparation was centrifuged for 2 hr. in Sorvall SW 27 rotor at 26,000 rpm. The purified membranes were washed twice with Hanks Buffered Saline Solution and finally with 25 mM Tris-HCl, pH 8.0 at 4°C
Detergent Solubilization of PI-glycan PLC. Freshly prepared rat liver plasma membranes were suspended in 20 ml 1% NP-40, 25 mM Tris-HCl buffer, pH 8.0, containing 20 ug/ml aprotinin and 10 ug/ml chymostatin and leupeptin. The suspension was homogenized in a loose-fitting teflon-glass homogenizer for 10 min. at 4°C and centrifuged for 1 hr. at 100,000 ×g. The resulting supernatant contained greater than 80% of PI-glycan PLC activity.
Assay of PI-glycan PLC Activity. Enzyme activity was assayed at 37° C. in a final volume of 1 ml with [3 H]myristate-labeled C-terminal glycopeptide in 25 mM Tris, pH 7.4, with 0.1% NP-40. The reaction was terminated after 1 hr. by extraction with 1 ml toluene. Organic and aqueous phases were separated by brief centrifugation at 100 ×g and the organic phase was taken to dryness in vacuo, redissolved in 40 ul of chloroform:methanol (2:1) containing 5 ug dimyristylglycerol, and spotted on a Silica G TLC plate which had been preactivated by baking at 125°C for 1-2 hr. The plate was developed twice in petroleum ether:diethyl ether:acetic acid (70:30:2) and then stained with iodine vapor to localize DAG. The DAG region was scraped and counted. Alternatively, 0.5 ml of the organic phase was taken directly for liquid scintillation counting.
Purification of PI-glycan PLC. The purification of PI-glycan PLC from rat liver plasma membranes is summarized in Table VIII. Purified liver plasma membranes were found to be highly enriched in PI-glycan-specific PLC activity, assayed by measuring the hydrolysis of the C-terminal glycopeptide of mfVSG. Activity was not detected in the cytosolic or nuclear fractions. Neutral nonionic detergents such as n-octylgluco-pyranoside and NP-40 were most effective at solubilizing enzyme activity. The zwitterionic detergent [3-(3-cholamidopropyl)-dimethylammonia]-1-propane sulfonate and the anionic detergent sodium deoxycholate were partially successful while cationic detergents either completely inhibited or failed to solubilize the activity (data not shown). Twenty ml of the solubilized enzyme were applied to a 20 ml DEAE-cellulose anion exchange column. Activity was eluted with buffer that contained 50 mM NaCl (FIG. 17). A broad peak of fractions which extracted radiolabel into the organic phase was observed. However, when analyzed by TLC only a few of the fractions exhibited significant DAG-releasing activity.
The DAG producing fractions were pooled and eluted through a column of Extractigel D of equal volume to remove detergent. PI-glycan PLC was then adsorbed onto a 10 ml column of butyl-agarose. The enzyme was specifically eluted with 0.1% NP-40 in 25 mM Tris-HCl buffer, pH 7.5 at 0°C Interestingly, total activity increased after this step, suggesting the removal of some inhibitory factor by butyl-agarose (FIG. 17, Table VIII). Analysis of the lipid products after digestion of the C-terminal glycopeptide of mfVSG with fractions of the partially purified enzyme (prior to butyl-agarose chromatography) showed free fatty acid production in adddition to DAG (data not shown). Contamination of this preparation with phospholipase A or diglyceride lipase activities could result in anomalously low PLC activity. PI-glycan PLC was purified 800 fold from purified plasma membranes. Seventy grams of rat liver yielded approximately 120 ug of enzyme. Taking into account yield of enzyme, this reflects a purification of approximately 20,000 over whole liver.
Samples from each stage of the purification were subjected to NaDodSO4 -PAGE in 10% gels. Silver staining of the enzyme after chromatography on butyl-agarose (FIG. 18, lane 4) revealed one major band with an apparent molecular weight of 52,000 daltons.
The molecular size of PI-glycan PLC was also evaluated on a 50 ml AcA 44 column, eluted in 0.5% NP-40 (FIG. 19). Activity was eluted with approximately 28 ml., indicating an apparent molecular size of 62,000 daltons with a Stokes radius of approximately 35 Angstroms. Comparison of the electrophoretic mobility and gel permeation profile of the enzyme indicates some association with detergent or lipid, but suggests that the catalytic activity of the enzyme is essentially monomeric in its active form.
PI-glycan PLC Catalyzes the Hydrolysis of Glycosylated-PI Substrates. The activity of the purified phospholipase was assayed using [3 H]inositol-labeled PI-glycan as substrate. The [3 H]-myristylated substrates were incubated with the enzyme and the lipid reaction products were extracted with toluene or chloroform:methanol (200:1) and analyzed on a neutral lipid system (FIGS. 21 and 22). Purified preparations of PI-glycan PLC catalyzed the cleavage of the phosphodiester bond and released DAG from both substrates.
The hydrolysis of the [3 H]inositol-labeled PI-glycan derived from BC3 Hl myocytes by the enzyme was evaluated by chromatography of the aqueous products (FIG. 9). The purified PI-glycan PLC was reconstituted by sonication of the HPLC purified PI-glycan in lipid vesicles containing 1:1 mixtures of phosphatidylcholine: phosphatidylethanolamine. Following a 2 hour incubation, the reaction was extracted and phase separated as described in the legend to FIG. 22, and the aqueous products were directly chromatographed on an HPLC anion exchange column. The enzyme caused the production of PI-glycan, eluting at 15 minutes. Further chemical and chromatographic evaluation of this product confirmed its identity as the insulin-sensitive enzyme modulator (data not shown).
Although PI-glycan PLC was active against mfVSG and the BC3 Hl derived PI-glycan, the enzyme exhibited no activity in hydrolysis of PI, PI-4,5-biphosphate, or phosphatidylcholine under a variety of reaction conditions, including variations in calcium concentration, thiol reagents, other phospholipids and additional cold substrate. These finding suggest the clear specificity of the enzyme for glycosylated phosphoinositides.
Characterization of PI-glycan PLC activity. The effects of cations and thiol reagents on the activity of the purified enzyme are summarized in Table IX. The presence of calcium was not required for enzyme activity, and the enzyme was stimulated by treatment with the chelator EGTA. Like the PI-glycan PLC purified from Trypanosoma brucei, the analogous activity from rat liver plasma membranes was activated by exposure to sulfhydryl reagents such as 2-mercaptoethanol, and inhibited by mercurial compounds. The activity is stable for approximately 1 week at 4°C or 1 month at -20°C Enzyme activity is stabilized by glycerol and ethylene glycol but is destroyed with repeated freeze-thawing.
| TABLE VIII |
| ______________________________________ |
| Purification of PI-glycan PLC |
| Specific |
| Total Total Activity |
| Protein Activity (pmol/ Purification |
| Yield |
| Step (mg) (pmol/min) |
| min/mg) |
| (Fold) (%) |
| ______________________________________ |
| detergent |
| 125 4.25 0.034 -- -- |
| solubilized |
| membranes |
| supernatant |
| 36 3.42 0.10 2.8 80 |
| after |
| centrifu- |
| gation |
| DE52 2.1 1.48 0.50 14.7 35 |
| Butyl- 0.12 3.18 26.5 780 76 |
| Agarose |
| ______________________________________ |
Samples from each stage of the purification were incubated in 1 ml with 25 nM [3H]mfVSG for 2 hr at 37°C and then extracted twice with 1 ml chloroform:methanol (200:1). The organic phases were taken to dryness and analyzed by TLC as described under Experimental Procedures. Each assay was performed in duplicate and was consistant within 10%. Specific actvity is expressed as pmol of mfvSG consumed/min/mg protein but does not reflect activities at saturating concentration of substrate.
| TABLE IX |
| ______________________________________ |
| Effects of Cations and Sulfhydryl Reagents of PI-glycan PLC |
| Addition Activity (dpm/2 hr) |
| % control |
| ______________________________________ |
| None 3970 100 |
| 100 uM EGTA 5900 148 |
| 100 uM EGTA, 100 uMCaCl2 |
| 6250 157 |
| 50 mM 2-mercaptoethanol |
| 7564 190 |
| 100 uM HgCl2 677 17 |
| ______________________________________ |
Purified PI-glycan PLC was incubated with 25 nM mfVSG in the presence of the indicated reagents for 2 hr at 37°C Enzyme activity was assayed as described for Table VIII. All assays were performed in duplicate variance was less than 10%.
This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
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